scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

scholarly journals Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1584-1589 ◽  
Author(s):  
Wenbin Li ◽  
Zonghe Yan ◽  
Mark K. Nakhla ◽  
Andrea M. Skantar
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
The United States ◽  
Grass Species ◽  
Specific Primers ◽  
Accurate Identification ◽  
Detection And Identification ◽  
Stem Gall ◽  
Species Specific

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

2004 ◽  
Vol 59 (1) ◽  
pp. 141-146 ◽  
Author(s):  
G.H. Reischer ◽  
M. Lemmens ◽  
A. Farnleitner ◽  
A. Adler ◽  
R.L. Mach
Keyword(s):  
Real Time ◽  
Fusarium Graminearum ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Species Specific

scholarly journals Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

2004 ◽  
Vol 70 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Kado ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Pcr Detection ◽  
Distribution Analysis ◽  
Specific Primers ◽  
Cell Counts ◽  
Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

scholarly journals Development of Primers and Probes for Genus and Species Specific Detection of ‘Candidatus Liberibacter Species’ by Real-Time PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1235-1243 ◽  
Author(s):  
G. Ananthakrishnan ◽  
N. Choudhary ◽  
Avijit Roy ◽  
V. G. Sengoda ◽  
E. Postnikova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Rrna Gene ◽  
Single Pair ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Genus Level ◽  
Candidatus Liberibacter ◽  
Species Specific

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different ‘Candidatus Liberibacter species’, ‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’, and ‘Ca. Liberibacter africanus’, which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of ‘Ca. Liberibacter’ at the genus level, providing increased convenience. Recent molecular analyses of ‘Ca. Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus ‘Ca. Liberibacter species’, enabling detection of ‘Ca. Liberibacter’ at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected ‘Ca. Liberibacter solanacearum’, a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

scholarly journals The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

2005 ◽  
Vol 51 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Simon D. Atkins ◽  
Ian M. Clark ◽  
Sonal Pande ◽  
Penny R. Hirsch ◽  
Brian R. Kerry
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Paecilomyces Lilacinus ◽  
Specific Primers ◽  
Species Specific

A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene

2020 ◽  
Vol 76 ◽  
pp. 102069
Author(s):  
Emilly Henrique dos Santos ◽  
Lidia Yamamoto ◽  
Wilson Domingues ◽  
Silvia Maria di Santi ◽  
Kelly Aparecida Kanunfre ◽  
...  
Keyword(s):  
Real Time ◽  
Cytochrome B ◽  
Real Time Pcr ◽  
Plasmodium Malariae ◽  
Cytochrome B Gene ◽  
Mitochondrial Cytochrome ◽  
Specific Primers ◽  
Mitochondrial Cytochrome B ◽  
Mitochondrial Cytochrome B Gene ◽  
Species Specific

scholarly journals Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilu-tion Assay, and Real-Time PCR Analysis

2020 ◽  
Author(s):  
Sepideh HAGHDOUST ◽  
Mahdieh AZIZI ◽  
Mostafa HAJI MOLLA HOSEINI ◽  
Mojgan BANDEHPOUR ◽  
Mandana MOHSENI MASOOLEH ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Parasite Burden ◽  
Pcr Analysis ◽  
Green Fluorescent ◽  
Limiting Dilution

Background: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. Methods: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. majorEGFP, and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. majorEGFP, and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. Results: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. majorEGFP was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). Conclusion: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.

Molecular diagnostics of the pigeon pea cyst nematode, Heterodera cajani Koshy, 1967, using real-time PCR

Nematology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Tatiana V. Roubtsova ◽  
Sergei A. Subbotin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pigeon Pea ◽  
Cyst Nematode ◽  
Taqman Probe ◽  
28S Rrna ◽  
Specific Primer ◽  
Pcr Assay ◽  
Species Specific ◽  
Heterodera Cajani

Summary The pigeon pea cyst nematode, Heterodera cajani, is an important nematode pest of pigeon pea that is present in all major growing regions of this crop in India and reported from Pakistan, Egypt and Myanmar. In this study, a new real-time PCR assay for detection of H. cajani using a species-specific primer and a TaqMan probe was developed. The primers and a probe were designed to amplify the COI gene fragment. The specificity of the primer-probe set was tested in singleplex or multiplex reactions against target and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer-probe sets, the signals were simultaneously observed for COI and D3 of 28S rRNA target genes. The results showed that the real-time PCR assay with species-specific primer and probe was sensitive enough to detect H. cajani DNA extracted from 0.003 egg or second-stage juvenile.

Sign in / Sign up

Export Citation Format

Share Document